[unreadable] Reactive oxidant species (ROS), such as nitric oxide (NO) and hydrogen peroxide (H2O2), play an important physiologic role in living tissue, and a pathophysiologic role in diseases, particularly those involving inflammation. Although cyclic nucleotides are key mediators of ROS-induced cellular processes, cyclic nucleotide-independent mechanisms are also important. In the previous grant cycle, we obtained data showing that the cyclic nucleotide-independent inhibition of smooth muscle contraction by ROS is due to novel mechanisms that inhibit Ca2+ sensitivity. This effect is spontaneously reversible in intact tissue and due to inhibition of the actomyosin ATPase activity of myosin II and the activities of myosin light chain kinase (MLCK) and heterotrimeric G-proteins. The overall goal of the current proposal is elucidate the biochemical mechanisms for redox regulation of these proteins by ROS. Aim A will test the hypotheses that ROS inhibit actomyosin ATPase activity by inhibiting nucleotide binding at the catalytic site and by stabilizing the myosin structure, thereby preventing F-actin binding. Both of these mechanisms are due to reversible oxidation of cysteine (Cys) residues on myosin. Aim B will test hypotheses related to ROS-induced inhibition of phosphorylation of the regulatory light chain of myosin (rMLC). Our preliminary data indicate that MLCK activity and GDP-GTP exchange at the Galpha subunit of heterotrimeric G-proteins are inhibited by ROS; both of these effects would inhibit rMLC phosphorylation. A permeabilized preparation is used for in situ biochemical studies, thereby demonstrating the physiologic relevance of the proposed mechanisms. Soluble proteolytic fragments of isolated myosin II and site-directed mutagenesis of candidate Cys on myosin are used to explore specific biochemical mechanisms for ROS effects on actomyosin ATPase activity. Purified MLCK holoenzyme and a constituitively active, proteolytic subfragment of the catalytic domain of MLCK are used to investigate biochemical mechanisms on MLCK. Finally, a crude membrane preparation and recombinant Gprotein subunits are used to elucidate novel redox effects on GDP-GTP exchange at Galpha. Elucidation of these mechanisms is of importance in understanding the role of ROS as key mediators of physiologic effects in both health and disease. [unreadable] [unreadable]